Wheat is one of the most important crops for food and feed and its straw is a potential feedstock for biorefinery purposes. Nitrogen (N) is an essential input factor in wheat agriculture but no information is available on how it affects straw composition during maturation and at harvest. To investigate this, we conducted a large scale field experiment in which wheat plants were cultivated at three levels of externally applied N. The plants were harvested at different stages of maturation, spanning green straw at heading (ear emergence) to fully yellow straw at final maturity. Defined parts of the straw were analyzed for cell wall characteristics relevant for further biomass processing. The straw N concentration corroborated with the level of N input, but the yield of straw biomass was not largely affected. High N treatment modified cell wall composition, namely increased abundance of arabinogalactan proteins (AGPs) and lignin in the mature straw. A general decrease in pectin methylesterification as well as in ferulate linkages was also observed. Importantly, no significant changes in crystalline cellulose and silicon concentration or in saccharification efficiency were observed among the different N treatments. Nitrogen fertilization partially alters the cell wall composition in wheat straw but is not a limiting factor in wheat biomass refinery.